Detection and identification of yeast and fungi as the cause of infections has never been more important. The numbers of immunocompromised patients at risk for yeast and fungal infection continues to increase, as does the spectrum of fungal agents causing disease. Mortality from fungal infections, particularly invasive fungal infections, is 30% or greater in certain at risk patient groups (“Stakeholder Insight: Invasive fungal infections”, Datamonitor, January 2004). The array of available antifungal agents is growing; however, so too is the recognition of both intrinsic and emerging resistance to antifungal drugs. These factors are contributing to the increased need for cost containment in laboratory testing and has led to laboratory consolidation in testing procedures.
Invasive fungal infections are on the increase. In 2003, it was estimated that there were 9 million at risk patients of which 1.2 million developed infection. Immunocompromised patients including transplant and surgical patients, neonates, cancer patients, diabetics and those with HIV/AIDs are at high risk of developing invasive fungal infections (Datamonitor report: Stakeholder opinion—Invasive fungal infections, options outweigh replacements 2004). A large number of severe sepsis are reported each year. Despite improvements in its medical management, sepsis still constitutes one of the greatest challenges in intensive care medicine. Micro-organisms (bacteria, fungi and yeast) responsible for causing sepsis are traditionally detected in hospital laboratories with the aid of microbiological culture methods with poor sensitivity (25-82%), which are very time-consuming, generally taking from two to five days to complete, and up to eight days for the diagnosis of fungal infections Definitive diagnosis is usually based on either the recovery and identification of a specific agent from clinical specimens or microscopic demonstration of fungi with distinct morphological features.
However, there are numerous cases where these methods fail to provide conclusive proof as to the infecting agent. In these instances, the detection of specific host antibody responses can be used, although again this can be affected by the immune status of the patient. Time is critical in the detection and identification of bloodstream infections typically caused by bacteria and fungi. Effective treatment depends on finding the source of infection and making appropriate decisions about antibiotics or antifungals quickly and efficiently. Only after pathogens are correctly identified can targeted therapy using a specific antibiotic begin. Many physicians would like to see the development of better in vitro amplification and direct detection diagnostic techniques for the early diagnosis of yeast and fungi (“Stakeholder Insight: Invasive fungal infections”, Datamonitor, January 2004). Recently Roche™ launched a real time PCR based assay (Septifast™), for the detection of bacterial, fungal and yeast DNA in clinical samples. Therefore there is a clear need for the development of novel rapid diagnostic tests for clinically significant bacterial and fungal pathogens for bioanalysis applications in the clinical sector. This has led us to the search and identify novel fungal and yeast nucleic acid targets for application in Nucleic Acid Diagnostisc (NAD) tests.
Candida spp. and Aspergillus spp. now rank as the most prominent pathogens infecting immunosupressed patients. In particular, infections are common in the urinary tract, the respiratory system and the bloodstream, at the site of insertion of stents, catheters and orthopaedic joints. Approximately, 10% of the known Candida spp. have been implicated in human infection. Invasive candidiasis occurs when candida enters the bloodstream and it is estimated to occur at a frequency of 8/100,000 population in the US with a mortality rate of 40%. Candida albicans is the 4th most common cause of bloodstream infection. Aspergillosis usually begins as a pulmonary infection that can progress to a life-threatening invasive infection in some patients and has a mortality rate of greater than 90%. Emerging mycoses agents include Fusarium, Scedosporium, Zygomycetes and Trichosporon spp. (“Stakeholder Insight: Invasive fungal infections”, Datamonitor, January 2004).
Fungal and yeast nucleic acid based diagnostics have focused heavily on the ribosomal RNA (rRNA) genes, RNA transcripts, and their associated DNA/RNA regions. The rRNA genes are highly conserved in all fungal species and they also contain divergent and distinctive intergenic transcribed spacer regions. Ribosomal rRNA comprises three genes: the large sub-unit gene (28S), the small sub-unit gene (18S) and the 5.8S gene. The 28S and 18S rRNA genes are separated by the 5.8S rRNA and two internal transcribed spacers (ITS1 and ITS2). Because the ITS region contains a high number of sequence polymorphisms, numerous researchers have concentrated their efforts on these as targets (Atkins and Clark, 2004). rRNA genes are also multicopy genes with >10 copies within the fungal genome.
A number of groups are working on developing new assays for fungal and yeast infections. US2004044193 relates to, amongst a number of other aspects, the transcription factor CaTEC1 of Candida albicans; inhibitors thereof, and methods for the diagnosis and therapy of diseases which are connected with a Candida infection; and also diagnostic and pharmaceutical compositions which contain the nucleotide sequences, proteins, host cells and/or antibodies. WO0183824 relates to hybridization assay probes and accessory oligonucleotides for detecting ribosomal nucleic acids from Candida albicans and/or Candida dubiniensis. U.S. Pat. Nos. 6,017,699 and 5,426,026 relate to a set of DNA primers which can be used to amplify and speciate DNA from five medically important Candida species. U.S. Pat. No. 6,747,137 discloses sequences useful for diagnosis of Candida infections. EP 0422872 and U.S. Pat. No. 5,658,726 disclose probes based on 18S rRNA genes, and U.S. Pat. No. 5,958,693 discloses probes based on 28S rRNA, for diagnosis of a range of yeast and fungal species. U.S. Pat. No. 6,017,366 describes sequences based on chitin synthase gene for use in nucleic acid based diagnostics for a range of Candida species.
It is clear though, that development of faster, more accurate diagnostic methods are required, particularly in light of the selection pressure caused by modern anti-microbial treatments which give rise to increased populations of resistant virulent strains with mutated genome sequences. Methods that enable early diagnosis of microbial causes of infection enable the selection of a specific narrow spectrum antibiotic or antifungal to treat the infection (Datamonitor report: Stakeholder opinion—Invasive fungal infections, options outweigh replacements 2004; Datamonitor report: Stakeholder Opinion-Sepsis, under reaction to an overreaction, 2006).
RPS7 is one of more than 70 ribosomal proteins. It is found in prokaryotes and eukaryotes and functions in the small ribosomal subunit in the folding of rRNA which forms the head of the small ribosomal subunit. The rps7 gene encodes an essential protein which has a conserved function within the ribosome. In yeasts, for example Saccharomyces cerevisiae RPS7 is encoded by two genes differing at 14 base pair positions with each gene having 1 intron. Synetos et al. (1992) showed that Saccharomyces could survive with one copy of the gene but that deletion of both was lethal. Delbrück et al. (1997) cloned and sequenced the rps7 gene in C. albicans (GenBank Accession number U37009), determining that rps7 in C. albicans lacked an intron and shared 83% homology at an amino acid level with the RPS7 protein in S. cerevisiae. This group also showed that the rps7 gene was up-regulated during hyphal formation with expression levels 3-6 fold higher than rRNA. This suggests that the gene is clinically relevant as morphogenesis from yeast form to hyphal formation is important in Candida spp. infections. In Aspergillus spp. in particular A. fumigatus, the rps7 gene contains 3 exons and 2 introns and therefore the structure of the gene is different from those found in yeasts.
It is therefore an object of the invention to provide sequences and/or diagnostic assays that may be used in detection and identification of one or more yeast or fungal species. The present inventors have exploited the structural organization of the rps7 gene to design Candida and Aspergillus gene-specific primers. This has an advantage over the prior art in that if one wants to identify a fungal pathogen in a sample which contains Candida as a commensal, the approach of using universal primers may not be successful. There is a strong possibility that the Candida will out-compete the fungal pathogen in the amplification process and will be preferentially amplified, resulting in failure to detect the disease-causing pathogen. Furthermore, it has been suggested by Delbrück et al. 1997 that the sequence differences between different alleles of the rps7 gene on different chromosomes in one species may be even greater than differences between genes in different related asexual species. This would lead the skilled person away from selecting this gene as a target for molecular diagnostics. Also, different sequence types exist for some species, such as Candida albicans, which would also lead one away from selecting this gene as a target gene for molecular diagnostics.